Fractional enrichment of proteins using [2- 13 C]-glycerol as the carbon source facilitates measurement of excited state 13 Cα chemical shifts with improved sensitivity
 

Fractional enrichment of proteins using [2- 13 C]-glycerol as the carbon source facilitates measurement of excited state 13 Cα chemical shifts with improved sensitivity

Abstract

A selective isotope labeling scheme based on the utilization of [2-13C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13Cα chemical shifts using Carrâ??Purcellâ??Meiboomâ??Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-13C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state 13Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06Â*ppm for exchange rates ranging from 100 to 1000Â*sâ??1, despite the small fraction of 13Cαâ??13Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using 13Cα spin probes.



Source: Journal of Biomolecular NMR